Streptococcus agalactiae (Group B streptococci, GBS) are p-hemolytic, gram-positive cocci that cause invasive infections in human neonates and adults. Despite significant advances in prevention and treatment of early onset neonatal disease, rates of GBS-related still births, prematurity, late onset neonatal disease and adult infections are not decreased or prevented. These observations emphasize the importance of alternate strategies for prevention of GBS infections. GBS encounters a wide array of host environments during its disease cycle. Hence, it is essential for the pathogen to rapidly adapt to changing external environments to survive and establish successful infections. Bacterial two component signaling (TCS) systems are critical for pathogens to mediate their adaptive responses to the external/host environment. A TCS composed of a sensor kinase CsrS and a response regulator CsrR regulate expression of GBS cytotoxins i.e. p-hemolysin (P-H/C) and CAMP factor. Our studies have shown that GBS also encodes two eukaryotic-type signaling enzymes comprising a sensor kinase Stk1 and its cognate phosphatase Stp1 that regulate virulence. This proposal seeks to extend our understanding of eukaryotic-type signaling in GBS. Our studies show that the eukaryotic-type kinase Stk1 regulates the expression of GBS cytotoxins (P-H/C and CAMP factor) in a manner that is opposite to the TCS, CsrR/CsrS. Our observation that Stk1 requires CsrR for regulation of cytotoxin expression demonstrates a link between eukaryotic-type and two-component signaling in GBS. The objective of this proposal is to elucidate the interaction between Stk1 and CsrR for adaptive gene expression in GBS. In this proposal, we will also define the role of the cognate signaling components Stp1 and CsrS that regulate Stk1 and CsrR activity, respectively. The three specific aims proposed will 1) define the role of Stp1 on Stk1 regulation of CsrR activity 2) establish the effect of the interaction between Stk1 and CsrR on the ability of CsrS to activate CsrR and 3) determine the functional consequence of Stk1 regulation of CsrR activity. A combination of genetic, molecular, biochemical and proteomic approaches are proposed to elucidate the link between eukaryotic-type and two component signaling mechanisms in GBS. These studies will broaden our understanding on signaling mechanisms employed by GBS to mediate its adaptive responses. This work will provide the foundation for future studies on adaptive gene expression by GBS for survival in various host niches and for evaluation of these signaling components as targets in antimicrobial strategies. Our findings will also have widespread implications on novel mechanisms of signaling that regulate adaptive responses in other pathogenic and non-pathogenic microbes.